CD31, also known as PECAM-1, is a transmembrane inhibitory receptor present on many kinds of haematopoietic cells. It is found on the surface of platelets, monocytes, neutrophils, T and B-cells and endothelial cells.
CD31 is composed of an extracellular domain (ECD) comprising 6 Ig-like domains numbered starting from the most distal from the membrane, a trans-membrane segment and a cytoplasmic tail containing 2 immuno tyrosine-based inhibitory motifs (ITIMs). The latter are not phosphorylated in resting conditions because CD31 does not possess an intrinsic kinase activity.
CD31 molecules on interacting cells bind to each other via a trans-homophilic interaction of the Ig-like domain 1 of the ECD which triggers the clustering of the protein through a cis-homophilic interaction of juxtamembrane sequences of the ECD, thus favoring the phosphorylation of the CD31 intracellular ITIMs by tyrosine kinases carried by other cluster-associated membrane receptors.
Binding of CD31 molecules on interacting cells thus triggers the phosphorylation of intracellular tyrosine inhibitory motifs and the recruitment and activation of SH2-containing phosphatases signalling pathways. Depending on the signalling adaptors associated to the closest membrane receptor, this can lead to either the activation of signalling cascades (e.g. GAB/ERK/MAPK, survival of endothelial cells) or their inhibition (e.g. JAK/STAT, activation of leukocytes and platelets).
The putative immunoregulatory properties of CD31 are supported by the fact that CD31 signaling drives mutual repulsion of blood leukocytes and modulates the balance between inhibitory and stimulatory signals of both innate and adaptive immune cells.
A soluble form of CD31 (‘splice variant’ form of CD31), produced from a variant transcript lacking the transmembrane segment, has been reported to be present in human plasma and is thought to be derived from endothelial cells (Goldberger al al 1994; J Biol Chem 269, 17183-17191). Soluble CD31 comprising the first Ig-like ECD domains can be immunodetected in body fluids by commercially available kits. However, studies attempting to find a correlation between plasma levels of soluble CD31 and the risk of atherothrombosis or other autoimmune diseases gave contradictory results as the data showed a broad range of plasma CD31 values, independently of the specific genetic polymorphisms analyzed.
A part of this puzzle was solved by the inventors' previous identification of an additional soluble form of CD31 derived from a portion of CD31 cleaved and shed by activated lymphocytes, as described in PCT/EP2009/058220 (WO2010/000756). It had long been observed that CD31 is “lost” on certain circulating lymphocytes and upon lymphocyte activation and the assumed absence of lymphocyte CD31 was increased in subjects suffering from atherothrombosis. The inventors found that the assumed loss of CD31 on activated/memory T lymphocytes is actually incomplete and results from the cleavage and shedding of CD31 between the 5th and the 6th extracellular Ig-like domains. The shed extracellular domain portion of CD31 (referred to as “lymphocyte-derived shed CD31”) is released into body fluids and can be detected in the circulation, where it is present together with the soluble splice variant form of CD31. They additionally showed that, after clinical symptoms disappear, high risk of recurrent atherothrombosis is linked with the specific increase of the lymphocyte-derived shed CD31 and the reciprocal decrease of the splice variant CD31 in the circulation. This provided a possible explanation of why immunodetection of the total circulating CD31 (comprising the lymphocyte-derived shed CD31 and the splice variant) by commercially available kits was not of diagnostic value.
This finding created the opportunity to establish diagnostic tools which could distinguish the different forms of soluble CD31. Previous commercially available tests detected plasma CD31 through the use of antibodies directed to CD31 domains 1 to 5, so could not discriminate between the soluble splice variant of CD31 (containing all the 6 extracellular Ig-like domains) and the lymphocyte-derived shed form of CD31 (containing domains 1 to 5 only). The method developed by the inventors and described in WO2010/000756 allowed the simultaneous detection of the 5th and of the 6th Ig-like domain on soluble CD31 captured from the 1st Ig-like domain. This method allowed the lymphocyte-derived shed form (which consists of the first 5 ECD domains and lacks the 6th Ig-like domain) to be precisely quantified and distinguished from CD31 forms that comprise the 6th Ig-Like domain (such as the described splice variant).
This finding also explained the absence of CD31 signalling on lymphocytes which have shed CD31. CD31 signalling is suppressed by the cleavage and shedding of the first 5 Ig-like domains of the ECD. Due to the loss of the “adhesive” Ig-like domains 1-2, the truncated CD31 cannot be bound by membrane anchored or the soluble splice variant forms of CD31, so CD31 signalling is absent on cells bearing the truncated CD31 molecule. This absence of CD31 signalling favours the activation of leukocytes and leads to pathologies linked to chronic tissue inflammation. The measure of lymphocyte-derived shed CD31 form could therefore reflect the individual risk of inflammatory and thrombotic pathologic processes